[go: up one dir, main page]

WO2004068973A2 - Traitement du tabac par pulverisation et/ou au sol visant a reduire tsnas - Google Patents

Traitement du tabac par pulverisation et/ou au sol visant a reduire tsnas

Info

Publication number
WO2004068973A2
WO2004068973A2 PCT/US2004/002546 US2004002546W WO2004068973A2 WO 2004068973 A2 WO2004068973 A2 WO 2004068973A2 US 2004002546 W US2004002546 W US 2004002546W WO 2004068973 A2 WO2004068973 A2 WO 2004068973A2
Authority
WO
WIPO (PCT)
Prior art keywords
tobacco
chemical solution
plant
antioxidants
leaves
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2004/002546
Other languages
English (en)
Other versions
WO2004068973A3 (fr
Inventor
Qinglin Li
Walter P. Hempfling
Marc R. Krauss
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Philip Morris Products SA
Original Assignee
Philip Morris Products SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Philip Morris Products SA filed Critical Philip Morris Products SA
Publication of WO2004068973A2 publication Critical patent/WO2004068973A2/fr
Publication of WO2004068973A3 publication Critical patent/WO2004068973A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/28—Treatment of tobacco products or tobacco substitutes by chemical substances
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/28—Treatment of tobacco products or tobacco substitutes by chemical substances
    • A24B15/30—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances

Definitions

  • the invention relates to methods for increasing the concentration of advantageous antioxidants in tobacco leaves by spraying at least one chemical solution onto a growing tobacco plant and/or soil treatment of growing tobacco plants.
  • the application of the chemical solution preferably occurs between topping and harvest, and can be optimized to stimulate the production of antioxidants in tobacco leaves which interfere with the formation of TSNAs during curing of the tobacco leaves.
  • Tobacco-specific nitrosamines such as N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK)
  • NNN N-nitrosonornicotine
  • NNK 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone
  • NNK 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone
  • TSNAs are not present in significant quantities in growing tobacco plants or fresh cut tobacco (green tobacco), but are formed during the curing process.
  • Bacterial populations which reside on the tobacco leaves are stated to largely cause the formation of nitrites from nitrate during curing and possibly effect the direct catalysis of the nitrosation of secondary amines at physiological pH values.
  • the affected secondary amines include tobacco alkaloids, which form TSNAs when nitrosated.
  • a method of reducing tobacco-specific nitrosamines in cured tobacco comprises raising the levels of antioxidants in tobacco prior to being cured, wherein the levels of antioxidants are raised by spraying a chemical solution onto an aerial part (i.e. , that part of the plant growing above ground) of a tobacco plant prior to harvesting and wherein the chemical solution effects an increase in the level of antioxidants in the tobacco plant.
  • the chemical solution preferably includes a plant growth hormone such as abscicic acid (ABA) or analog thereof, a plant activator such as salicylic acid (SA) (commercially available as ACTIGARD®) or analog thereof, a plant activator such as a harpin protein containing product commercially available as MESSENGER®, a herbicide such as methyl viologen (MV) or analog thereof, a stress inducing agent such as hydrogen peroxide, sodium chloride (NaCl), or sulfur dioxide, or combinations thereof.
  • the analog of MV is Paraquat (a quaternary nitrogen herbicide widely used for weed control).
  • the tobacco plants are sprayed with the chemical solution in a single application or multiple applications about 1-3 weeks prior to harvest.
  • the tobacco is bur ley tobacco and the method further includes air curing the bur ley tobacco.
  • the tobacco is preferably incorporated in a cigarette.
  • a method of reducing tobacco-specific nitrosamines in cured tobacco comprises raising the levels of antioxidants in tobacco prior to being cured, wherein the levels of antioxidants are raised by soil treatment of tobacco plants prior to harvesting and wherein the soil treatment effects an increase in the level of antioxidants in the tobacco plant.
  • the soil can be treated with a chemical solution which preferably includes a plant growth hormone such as abscicic acid (ABA) or analog thereof, a plant activator such as salicylic acid (SA) (commercially available as ACTIGARD®) or analog thereof, a plant activator such as a harpin protein containing product commercially available as MESSENGER ® , a herbicide such as methyl viologen (MV) or analog thereof, a stress inducing agent such as hydrogen peroxide, sodium chloride, or sulfur dioxide, or combinations thereof.
  • the analog of MV is Paraquat (a quaternary nitrogen herbicide widely used for weed control).
  • the soil is treated in a single application or multiple applications about 1-3 weeks prior to harvest.
  • the tobacco is burley tobacco and the method further includes air curing the bur ley tobacco.
  • the tobacco is preferably incorporated in a cigarette.
  • Figure 1 depicts a graph of antioxidant capacity of green and cured Oriental, bright and burley tobacco grown without a spray treatment to raise the antioxidant capacity.
  • Figure 2 depicts a mechanism by which antioxidants in tobacco inhibit TSNA formation.
  • Figure 3 depicts a graph of the effects on antioxidant capacity of spraying salicylic acid and methyl viologen onto greenhouse tobacco.
  • the amount of tobacco specific nitrosamines (TSNAs) in cured tobacco leaves can be reduced by spraying growing tobacco plants with a chemical solution and/or treating the soil surrounding roots of growing tobacco plants with a chemical solution.
  • TSNAs tobacco specific nitrosamines
  • TSNAs in cured tobacco leaves may be reduced by raising the levels of antioxidants in the tobacco leaves prior to harvesting the plants.
  • the antioxidant levels in tobacco leaves sprayed with the chemical solution are preferably raised at least 25 % , preferably at least 30% , and more preferably at least 50% compared to tobacco leaves of untreated plants grown without the spray treatment.
  • the antioxidant levels in tobacco leaves of tobacco plants grown in soil treated with the chemical solution are preferably raised at least 25%, preferably at least 30%, and more preferably at least 50 % compared to tobacco leaves of untreated plants which without the soil treatment.
  • Figure 1 shows an example of the antioxidant capacity of green and cured Oriental, bright and burley tobacco obtained from untreated plants.
  • TSNAs are formed predominantly during the curing process.
  • TSNAs in cured tobacco leaves results from the reaction of a reactive nitrosating species with the tobacco alkaloids.
  • the amount of antioxidants in tobacco leaves during the time of air-curing is believed to be advantageous to the inhibition of TSNA formation.
  • An elevated concentration of native antioxidants during senescence and air-curing of tobacco can be obtained by spraying of a chemical solution preferably comprising an aqueous solution containing one or more chemical compounds onto the growing tobacco plant and/or treating the soil surrounding roots of growing tobacco plants with the aqueous solution.
  • a chemical solution preferably comprising an aqueous solution containing one or more chemical compounds onto the growing tobacco plant and/or treating the soil surrounding roots of growing tobacco plants with the aqueous solution.
  • it is possible to increase foliar contents of native antioxidants by stressing the tobacco plant, e.g. , using ABA to reduce the availability of carbon dioxide necessary for photosynthesis.
  • the tobacco is burley tobacco.
  • the spraying of the solution and/or soil treatment preferably occurs between topping (i.e.
  • the level of antioxidants is increased to an amount sufficient to prevent significant nitrosation during the yellowing and browning phases of curing.
  • the chemical spraying and/or soil treatment can be carried out only once or the tobacco plants can be sprayed and/or subjected to soil treatment more than once.
  • the plants can be sprayed at layby (when the plants are about knee-high) and the spray treatment can be repeated periodically such as every 5 to 15 days until harvest.
  • the soil in which the plants are grown can be treated at layby and the soil treatment can be repeated periodically such as every 5 to 15 days until harvest.
  • the spray and/or soil treatment can be combined with other methods of elevating tobacco leaf antioxidants to a level that inhibits nitrosation.
  • the antioxidants can be raised by pruning of the tobacco plants (e.g. , root pruning and xylem cutting) prior to harvest.
  • root pruning and/or xylem cutting may be used to inhibit TSNA formation during air-curing.
  • the plant's capacity to assimilate CO2 is reduced, and the photosynthetic electron flux to O2 will increase resulting in the increased production of superoxide and hydroxyl radicals.
  • These molecules induce antioxidants to cope with the oxidative stress.
  • Disturbances in photosynthetic activity can be used to cause the formation of reactive oxygen species.
  • chemicals that directly affect chloroplast activity can stimulate processes that induce formation of reactive oxygen species.
  • Redox-active herbicides such as the diphenyl ethers (e.g. , acifluorfen) can act through the production of reactive oxygen.
  • Such herbicides can cause the activity of antioxidants such as ascorbate, glulathione and glutathione reductase to increase while the plant is actively defending itself against the herbicide.
  • Bipyridyl herbicides such as Paraquat (also known as methyl viologen) and Diquat, can be used to increase oxidative stress directly by generating reactive oxygen radicals.
  • Growing tobacco plants can be sprayed with a chemical solution, preferably an aqueous solution, which effects an increase in antioxidant levels in the tobacco leaves and thus reduced TSNAs during curing of the tobacco leaves.
  • the soil of tobacco plants can be treated with a chemical solution, preferably an aqueous solution, which effects an increase in antioxidant levels in the tobacco leaves and thus reduced TSNAs during curing of the tobacco leaves.
  • a chemical solution containing at least one chemical compound is sprayed onto growing tobacco plants using farm equipment suitable for spray application of liquids to tobacco plants. If desired, spraying of such compounds can be combined with other nitrosamine-reducing treatments such as root pruning and xylem cutting, to result in tobacco leaves with exceptionally low nitrosamine contents during the curing process.
  • the soil surrounding the roots of tobacco plants can be sprayed and/or irrigated using a chemical solution which effects the desired increase in the antioxidant levels in the tobacco leaves.
  • herbicide is meant to include reagents that increase antioxidant activity.
  • preferred herbicides include but are not limited to redox-active herbicides and bipyridyl herbicides.
  • Preferred redox-active herbicides include diphenyl ethers (e.g., acifluorfen and acifluorfen sodium).
  • Preferred bipyridyl herbicides include methyl viologen (e.g. , Paraquat) and Diquat.
  • plant growth hormone is meant to include those plant growth hormones which modulate the antioxidant level and thereby TSNA in the tobacco plant.
  • Preferred plant growth hormones are abscicic acid (ABA) and jasmonic acid.
  • plant activator is meant to include reagents which modulate the antioxidant level and thereby TSNA in tobacco plants.
  • a preferred plant activator is a salicylic acid, or an analog of salicylic acid, (e.g. , ACTIGARD ® ), or a harpin protein (e.g. , MESSENGER ® ).
  • stress inducing reagents is meant to include reagents which modulate the antioxidant level and thereby TSNA in tobacco.
  • Preferred stress inducing reagents include sulfur dioxide, sodium chloride and hydrogen peroxide.
  • One embodiment comprises spraying an effective amount of at least one herbicide such as methyl viologen (MV) or one or more analogs thereof (e.g., Paraquat), or Diquat (chemical name: l' ,r-ethylene-2,2'-bipyridyldiylium) and/or other herbicides, to the aerial part of topped tobacco plants several weeks or days before harvesting. Also contemplated are diphenyl ethers.
  • MV methyl viologen
  • Paraquat Paraquat
  • Diquat chemical name: l' ,r-ethylene-2,2'-bipyridyldiylium
  • Preferred diphenyl ethers include but are not limited to acifluorfen and acifluorfen sodium, which respectively have the chemical names of 5- [2-chloro-4-(trifluoro-methyl)phenoxy] -2-nitrobenzoate and sodium 5-[2-chloro-4-(trifluoro-methyl)phenoxy]-2-nitrobenzoate.
  • the spraying is performed one or more times in the field about 1-3 weeks before harvesting (i.e. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting). It has been found that such spraying results in a significant reduction of TSNAs during air-curing of burley tobacco.
  • TSNAs during curing may be accompanied by an increase in total antioxidant activity, as measured by the Ferric- Reducing Antioxidant Power assay ("FRAP").
  • Another embodiment comprises applying an effective amount of at least one herbicide such as methyl viologen (MV) or one or more analogs thereof, such as Paraquat (chemical name: l,r-dimethyl-4,4'-bipyridinium), and/or other herbicides, to the soil surrounding roots of topped tobacco plants several weeks or days before harvesting.
  • the soil treatment is performed one or more times in the field about 1-3 weeks before harvesting (i.e. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting).
  • the herbicide(s) can be applied in a concentration of about 0.01 mM to about 0.3 mM, more preferably 0.05 to about 0.15 mM and most preferably in an amount of about 0.08 to about 0.11 mM (e.g. , about 0.1 mM Paraquat) (and every 0.01 mM unit inbetween these ranges).
  • about 50 mL to about 500 mL of the solution is sprayed per plant, more preferably an amount of about 100 mL to about 300 mL and most preferably about 150 mL to about 250 mL of solution is sprayed onto the leaves or the soil of each plant.
  • Another embodiment comprises spraying an effective amount of a plant activator, such as salicylic acid (SA), or one or more analogs thereof, preferably ACTIGARD® (active ingredient chemical name: l,2,3-benzothiadiazole-7-thiocarboxylic acid-5-methyl-ester), or a harpin protein such as MESSENGER® (harpin, HrpN from Erwinia amylovora; GenBank Accession No: AAC31644), to, for example, the aerial part of topped tobacco plants several weeks or days before harvesting.
  • ACTIGARD® is manufactured by Syngenta Crop Protection, Greensboro, North Carolina.
  • MESSENGERTM is available from Eden Bioscience Corporation, Bothell, Washington.
  • the spraying is performed one or more times in the field about 1-3 weeks before harvesting (i.e. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting).
  • ACTIGARD® can be sprayed onto the tobacco plants in a single application at a concentration of about 1 ounce (oz) per acre about 1 week prior to harvest or in a series of applications at the same or different concentrations, e.g. , 1 oz/acre, 0.5 oz/acre and/or 0.25 oz/acre.
  • MESSENGER® can be sprayed onto the tobacco plants in a single application at a concentration of 9 ounces per acre or in a series of applications at the same or different concentrations.
  • ACTIGARD ® is believed to disable enzyme activity thereby creating oxidative stress to which the tobacco plant responds by production of antioxidants.
  • MESSENGER ® is believed to create a plant response known as systemic acquired resistance that manifests a production of antioxidants. Due to the increase in antioxidant level in the treated tobacco leaves, a significant reduction of TSNAs can be obtained during curing of the tobacco leaves.
  • a further embodiment comprises applying an effective amount of salicylic acid (SA), or one or more analogs thereof, preferably ACTIGARD ® , or a harpin protein such as MESSENGER®, to the soil surrounding roots of topped plants several weeks or days before harvesting.
  • SA salicylic acid
  • the soil treatment is performed one or more times in the field about 1-3 weeks before harvesting (i.e. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting).
  • Salicylic acid and its analogs such as ACTIGARD® can be used to treat plants in concentrations of about 0.1 mM to about 10 mM, or more preferably about 0.5 mM to about 2 mM and most preferably in an amount of about 0.8 mM to about 1.2 mM.
  • concentrations of about 0.1 mM to about 10 mM, or more preferably about 0.5 mM to about 2 mM and most preferably in an amount of about 0.8 mM to about 1.2 mM.
  • about 50 mL to about 500 mL of the solution is sprayed per plant, more preferably an amount of about 100 mL to about 300 mL and most preferably about 150 mL to about 250 mL of solution is sprayed onto the leaves of each plant or administered onto the soil surrounding the roots of each plant.
  • a harpin protein such as MESSENGER® can be used to treat the leaves or roots of the plants in an amount of about 4.5 oz/acre to about 9 oz/acre (9 oz/acre is the FDA approved amount).
  • Another embodiment of the present invention comprises spraying an effective amount of at least one plant growth hormone such as abscicic acid (ABA), or one or more of its more stable analogs, or jasmonic acid or one or more analogs thereof preferably onto the aerial part of topped tobacco plants several weeks or days before harvesting.
  • the spraying is performed one or more times in the field about 1-3 weeks before harvesting (i.e. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting). Due to the increase in antioxidant level in the treated tobacco leaves, a significant reduction of TSNAs can be obtained during curing of the tobacco leaves.
  • ABA is a major controller of stomatal closing, and naturally increases in concentration in tobacco leaves as a response to water-deficit stress. See Figure 2.
  • the CO2 supply for photosynthesis can be interrupted leading to enhancement of the native leaf antioxidants.
  • a solution of ABA, or a suitable analog can be sprayed onto the tobacco plant such that stomata remain at least partially closed for a short time period, but then open again to support photosynthesis, akin to the action of root pruning, but without loss of turgor due to water deficit.
  • the effects of spraying ABA on growing tobacco plants as compared to MV is shown in Figure 3.
  • an amount of about 0.05 mM to about 1 mM ABA is used, more preferably about 0.1 to about 0.5 mM ABA and most preferably about 0.2 mM to about 0.4 mM ABA.
  • Jasmonic acid is preferably administered to the leaves or the soil surrounding the roots of the tobacco plants in an amount of about 0.01 mM to about 2.0 mM and more preferably in an amount of about 0.1 mM to about 1 mM.
  • a further embodiment of the present invention comprises applying an effective amount of at least one plant growth hormone such as abscicic acid (ABA), or one or more of its more stable analogs or jasmonic acid or one or more analogs thereof onto the soil surrounding roots of topped tobacco plants several weeks or days before harvesting.
  • plant growth hormone such as abscicic acid (ABA)
  • ABA abscicic acid
  • the soil treatment is performed one or more times in the field about 1-3 weeks before harvesting (i.e. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting).
  • Stress Inducing Agent Another embodiment of the present invention comprises spraying a solution containing an effective amount of at least one stress inducing agent such as hydrogen peroxide, NaCl (salt) or sulfur dioxide onto the aerial part (the leaves) of topped tobacco plants several weeks or days before harvesting. Preferably, the spraying is performed one or more times in the field about 1-3 weeks before harvesting (i.e. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting).
  • a further embodiment of the present invention comprises applying a solution containing an effective amount of at least one stress inducing agent such as hydrogen peroxide, a salt (e.g.
  • the soil treatment is performed one or more times in the field about 1-3 weeks before harvesting (i.e. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting).
  • Hydrogen peroxide treatment is preferably applied as a solution containing hydrogen peroxide in a range of about 1 % to about 5 % , more preferably in a range of about 2% to about 4%, and most preferably in a range of about 2.5% to about 3.5% being sprayed on the leaves and/or onto the soil surrounding the roots.
  • NaCl is administered to the plant by spraying on the leaves or onto the soil surrounding the roots a solution of sodium chloride comprising about 0.8 % to 0.1 % sodium chloride, more preferably from about 0.6% to about 0.2% NaCl, and most preferably 0.5% to about 0.3 % (e.g. , about 0.4%) NaCl.
  • the sulfur dioxide is administered to the plant in an amount of about 50 nL/L to about 500 nL/L, more preferably in an amount of about 100 nL/L to about 250 nL/L and most preferably in an amount of about 150 nL/L to about 200 nL/L.
  • about 50 to about 500 mL of a solution with one or more of these reagents is sprayed per plant, more preferably an amount of about 100 to about 300 mL and most preferably about 150 to about 250 mL of solution is sprayed onto each plant.
  • reagents for example, one or more herbicides, stress inducing reagents, plant growth hormones, and plant activators can be combined either in an admixture or applied separately.
  • the combination of reagents can be used to treat the tobacco such that the tobacco has increased antioxidant activity and/or production of antioxidants, which correspondingly reduces TSNAs.
  • Combinations of such reagents include a plant growth hormone in combination with a plant activator (e.g. , a salicylic acid combined with jasmonic acid). Additional combinations include jasmonic acid in combination with one or more stress reducing reagents, one or more herbicides, one or more plant activators, or with a different plant growth hormone.
  • the combinations of reagents may require less of each reagent to be administered to the tobacco plant than if the reagent was administered alone (i.e., not in combination with other antioxidant inducing reagents).
  • Such combinations can be administered onto the aerial part of a tobacco plant (topped or prior to being topped).
  • the combination of reagents can be administered several weeks or days before harvesting, or both.
  • the combination of reagents are applied by spraying.
  • Such spraying can be performed one or more times in the field.
  • the spraying of the combination of reagents occurs about 1-3 weeks before harvesting (i.e. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting).
  • the combination of reagents can also be administered to the plant, or portions of the plant (e.g. , leaves, stems), after the plant has been harvested.
  • a further embodiment of the present invention comprises applying a solution containing an effective amount of a combination of reagents onto the soil surrounding the roots of a tobacco plants (topped or prior to topping).
  • the solution can be applied several weeks or days before harvesting the plant.
  • the soil treatment is performed one or more times in the field about 1-3 weeks before harvesting (i.e. , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before harvesting).
  • the solution can also be administered to the plant after the plant has been harvested.
  • about 50 to about 500 mL of the solution is sprayed per plant, more preferably an amount of about 100 to about 300 mL and most preferably about 150 to about 250 mL of solution is sprayed onto each plant.
  • the reagents discussed above can be used alone to reduce TSNAs or in combination with one another, or in combination with methods which reduce TSNAs.
  • the methods of treating the soil surrounding the roots or the leaves of the plants with one or more chemical compounds before harvesting can be combined with other methods of increasing antioxidants, to reduce nitrosamines in tobacco plants during the curing process.
  • partial removal of the root structure (root pruning) or xylem cutting of burley tobacco in the field about a week before harvesting, results in significant reduction of tobacco-specific nitrosamines during air-curing, probably due to the resulting increase in total antioxidant capacity (measured by the Ferric-Reducing Antioxidant Power assay) that persists during the curing process.
  • the root pruning (or other mechanical stress which produces a similar result) induces water-deficit stress which is believed to result in temporary stomatal closure to preserve water in the plant.
  • Such closure of the stomata also shuts off the supply of atmospheric carbon dioxide that supports photosynthetic carbon dioxide fixation and allows the plant to produce more antioxidants.
  • Other forms of mechanical stress which yield a similar result i.e. , induce production of antioxidants such as xylem cutting
  • TSNA Inhibition Using Salicylic Acid or Methyl Viologen Topped greenhouse tobacco plants were sprayed with aqueous solutions of salicylic acid, methyl viologen and a control substance, in order to measure the total antioxidant capacity ( ⁇ mol/g) over 24 hours. Mature burley tobaccos (with about 20-22 leaves per plant) were topped two weeks prior to being sprayed with either salicylic acid or methyl viologen. An aqueous solution of salicylic acid (99%; Aldrich Chemical Co.) at 1 M and methyl viologen (98%; Aldrich Chemical Co.) at 0.1 mM concentration were sprayed upon the tobacco leaves.
  • Each plant was sprayed with at least 200 mL of solution with good coverage of each leaf to produce a more uniform response to the chemical spraying. Green samples were taken 24 hours after spraying. Leaves were checked after sampling to make sure all leaves are representative of the plant conditions. Samples were quickly separated into lamina and midribs as known in the art. Only the lamina was retained for analysis. Sampled green lamina was flash-frozen in liquid nitrogen. The samples can be stored at - 80 °C if analysis cannot be performed immediately.
  • Total antioxidant capacity was determined by FRAP assay, and the values were expressed as ⁇ mol/g of fresh weight.
  • concentration of chlorogenic acid and rutin, two water soluble antioxidants, were measured by HPLC and their contribution to the total antioxidant capacity (measured by FRAP) was calculated.
  • the results (Table 1) show that both methyl viologen and salicylic acid increase antioxidant capacity.
  • the FRAP assay was performed as follows. The green lamina were accurately weighed and extracted with 10% methanol and 0.2 M perchloric acid in water. The mixture was shaken at 4°C on an orbital shaker for an hour. The extracts were then centrifuged at 5,000 rpm for 20 minutes. The supernatant was filtered through Whatman Autovial 0.45 ⁇ m PVDF syringe filters and was ready for analysis after appropriate dilution with extraction solution (10% MeOH and 0.2 M perchloric acid in water).
  • the procedures for the FRAP assay on tobacco samples were modified from that previously described by Benzie and Strain, Meth. Enzymology 299: 15-27 (1999). The assay was done manually on a spectrophotometer at room temperature.
  • the FRAP reagent was prepared by combining 300 mM acetate buffer (pH 3.6), 10 mM 2,4,5-tripyridyl-s- triazine in 40 mM HC1 and 20 mM FeCb in the ratio of 10:1: 1 (v:v:v). A 100 ⁇ L aliquot of the sample extraction was added to 3 mL of FRAP reagent and mixed. After the mixture stood at room temperature for 6 min.
  • the absorbance at 593 nm was determined against the FRAP reagent alone. Calibration was against a standard curve (i.e. , 500, 1000 and 2000 ⁇ M ferrous iron) produced by the addition of freshly prepared ferrous sulfate. FRAP values were calculated as micromolar ferrous ion (ferric reducing power) from two determinations.
  • Chlorogenic acid i.e. , 3-0-caffeoylquinic acid
  • rutin i.e. , quercetin 3- rhamnosidoglucoside
  • HPLC high performance liquid chromatographic
  • Other polyphenols such as the isomers of chlorogenic acid (i.e. , 4-0-, and 5-0-caffeoylquinic acid) and scopoletin (i.e. , 6- methoxy-7-hydroxycoumarin) were also separated by reverse phase HPLC using a gradient of methanol with 1 % glacial acetic acid.
  • Example 2 The following table (Table 2) shows the results of experiments using an aqueous chemical spray to raise burley native leaf antioxidant capacity in order to interfere with TSNA production during air curing.
  • Burley tobacco plants were grown using standard agronomic practices.
  • ACTIGARD® and MESSENGER® were sprayed on the plants after layby in two-week intervals with a total of five (5) applications of the reagents until the plants were harvested.
  • Treatment with the ACTIGARD® or MESSENGER® at this time in plant growth is not suitable for optimum results for the manufacturer recommended uses of those reagents as plant activators.
  • care was taken to ensure clean tobacco by minimizing contact with the soil.
  • Plants were stalk cut and five stalks were speared on each stick for standard curing in conventional air-curing barns. All treated and control tobaccos were hung with even spacing of sticks. The treated (non-control) tobaccos were surrounded by clean filler tobacco to help to create normal curing conditions. At least three replicate samples were taken at each sampling point from five different plants with 3 leaves from the top one-third of the plant (excluding the top 4 leaves). Each plant was sampled only once. Midveins were separated from the lamina. Both lamina and midveins were weighed and placed in sample bags stored at -80 °C. After freeze-drying, samples were re-weighed to determine the moisture content.
  • FRAP analysis was performed as follows. Freeze-dried samples were accurately weighed and extracted with 10% methanol and 0.2 M perchloric acid in water. The mixture was shaken at 4 °C on an orbital shaker for an hour. The extracts were then centrifuged at 5,000 rpm for 20 minutes. The centrifuged supernatants were then filtered through Whatman Auto vial 0.45 ⁇ m PVDF Syringe filters. The filtered material was then diluted as discussed in Example 1 above. The FRAP assay was conducted as described by Benzie and Strain (1999) with the following modifications.
  • the FRAP reagent was prepared daily by combining 300 mM acetate buffer (pH 3.6), 10 mM 2,4,5-tripyridyl-s- triazine in 40 mM HC1 and 20 mM FeCb in a ratio of 10:1: 1 (v:v:v).
  • a 100 ⁇ L aliquot of the sample extraction was added to 3,000 ⁇ L (3 mL) of FRAP reagent and mixed. After the mixture stood at room temperature for 6 minutes, the absorbance at 593 nm was determined against the FRAP reagent. Calibration was against a standard curve (i.e. , 500, 1000 and 2000 ⁇ M ferrous ion) produced by the addition of freshly prepared ferrous sulfate.
  • FRAP values were calculated as micromolar ferrous ion (ferric reducing power) from two determinations. The total antioxidant capacity values are expressed in ⁇ mol/g of fresh weight.
  • Example 3 NaCl Induced Inhibition of TSNAs
  • antioxidants interfere with the nitrosation of secondary alkaloids.
  • the amount of antioxidants present at the time of air-curing the tobacco plants is believed to be effective in inhibiting TSNA formation.
  • the objective is to increase the foliar concentration of native antioxidants during senescence and air-curing of burley tobaccos by judicious application of reagents which inhibit TSNAs.
  • application preferably occurs between topping of the tobacco plant and harvest.
  • NaCl sodium chloride

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Forests & Forestry (AREA)
  • Ecology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Cultivation Of Plants (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention concerne des procédés pour réduire la concentration d'antioxydants pendant la sénescence et le fanage des feuilles de tabac, qui comprennent l'application d'une solution chimique. Les procédés consistent à pulvériser la solution sur un plant de tabac avant la récolte et/ou appliquer la solution chimique au sol entourant les racines de plants de tabac en croissance. L'application de la solution chimique a lieu de préférence entre l'étêtage et la récolte; elle est optimisée pour stimuler la production d'antioxydants et intervenir dans la formation de TSNAs pendant le fanage. Le tabac peut être un tabac de type Burley, soumis au fanage à l'air.
PCT/US2004/002546 2003-01-31 2004-01-30 Traitement du tabac par pulverisation et/ou au sol visant a reduire tsnas Ceased WO2004068973A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US44384803P 2003-01-31 2003-01-31
US60/443,848 2003-01-31

Publications (2)

Publication Number Publication Date
WO2004068973A2 true WO2004068973A2 (fr) 2004-08-19
WO2004068973A3 WO2004068973A3 (fr) 2005-06-23

Family

ID=32850803

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/002546 Ceased WO2004068973A2 (fr) 2003-01-31 2004-01-30 Traitement du tabac par pulverisation et/ou au sol visant a reduire tsnas

Country Status (2)

Country Link
US (3) US20050034365A1 (fr)
WO (1) WO2004068973A2 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007053097A1 (fr) 2005-11-07 2007-05-10 Njette Ab Nicotine a teneur reduite en nitrosamines
US7757697B2 (en) * 2005-12-22 2010-07-20 Swisher International, Inc. Method for reducing nitrosamines in tobacco
WO2011042166A1 (fr) * 2009-10-09 2011-04-14 Philip Morris Products S.A. Traitement combiné d'un extrait de tabac utilisant des antioxydants et des épurateurs d'antioxydants
US7992575B2 (en) 2005-02-28 2011-08-09 U.S. Smokeless Tobacco Company Use of chlorate, sulfur or ozone to reduce tobacco specific nitrosamines
WO2013035505A1 (fr) * 2011-09-05 2013-03-14 日本たばこ産業株式会社 Procédé pour empêcher une augmentation des nitrosamines spécifiques au tabac pendant le stockage
WO2014024020A1 (fr) * 2012-08-08 2014-02-13 Gruppo Mauro Saviola S.R.L. Procédé de fabrication d'un tabac à basses teneurs en nitrosamines
CN104432463A (zh) * 2014-10-29 2015-03-25 湖北中烟工业有限责任公司 白肋烟加工醇化调香的方法
EP2981182A1 (fr) * 2013-04-05 2016-02-10 R. J. Reynolds Tobacco Company Modification du profil bactérien du tabac
US9271524B1 (en) 2007-09-07 2016-03-01 U.S. Smokeless Tobacco Company Tobacco having reduced tobacco specific nitrosamine content
CN105794477A (zh) * 2016-04-11 2016-07-27 广东省烟草南雄科学研究所 一种提高贫氯烟田烟叶氯离子含量的方法
CN107920582A (zh) * 2015-05-14 2018-04-17 R.J.雷诺兹烟草公司 烟草的处理
CN109699650A (zh) * 2019-01-29 2019-05-03 中国热带农业科学院环境与植物保护研究所 一种高效诱导荔枝产生抗病性的组合物
CN111011032A (zh) * 2019-12-11 2020-04-17 中国烟草总公司郑州烟草研究院 一种降低氯敏感作物中氯离子含量的方法
CN111693650A (zh) * 2020-06-10 2020-09-22 河南中烟工业有限责任公司 一种改善烤烟上六片烟叶质量的方法

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050034365A1 (en) * 2003-01-31 2005-02-17 Philip Morris Usa Inc. Spray and/or soil treatment of tobacco to reduce TSNAs
CA2872521C (fr) 2005-02-28 2017-04-18 22Nd Century Limited, Llc Reduction de niveaux d'alcaloides nicotiniques dans des plantes
PL2450446T3 (pl) 2006-09-13 2015-10-30 22Nd Century Ltd Llc Zwiększanie poziomów alkaloidów nikotynowych
US9551003B2 (en) 2006-09-13 2017-01-24 22Nd Century Limited, Llc Increasing levels of nicotinic alkaloids in plants
US9102948B2 (en) 2006-11-17 2015-08-11 22Nd Century Limited, Llc Regulating alkaloids
CN103005699A (zh) * 2012-12-13 2013-04-03 湖北中烟工业有限责任公司 提高烟叶钾含量的方法
US9980509B2 (en) * 2013-04-05 2018-05-29 R.J. Reynolds Tobacco Company Modification of bacterial profile of tobacco
US20150068544A1 (en) * 2013-09-09 2015-03-12 R.J. Reynolds Tobacco Company Smokeless tobacco composition incorporating a botanical material
US11503853B2 (en) * 2013-09-09 2022-11-22 R.J. Reynolds Tobacco Company Smokeless tobacco composition incorporating a botanical material
US10111458B1 (en) 2014-05-16 2018-10-30 R.J. Reynolds Tobacco Company Process for inhibiting formation of nitrosamines
US10721957B2 (en) 2016-10-04 2020-07-28 R.J. Reynolds Tobacco Company Tobacco-derived colorants and colored substrates
US10647989B2 (en) * 2016-10-07 2020-05-12 Altria Client Services Llc Composition and methods for producing tobacco plants and products having reduced tobacco-specific nitrosamines (TSNAs)
US11278050B2 (en) 2017-10-20 2022-03-22 R.J. Reynolds Tobacco Company Methods for treating tobacco and tobacco-derived materials to reduce nitrosamines
CN109288110B (zh) * 2018-09-10 2021-06-01 云南省烟草农业科学研究院 一种基于水杨酸施用缓解灰色烟的烤烟管理及烘烤方法
GB201900940D0 (en) 2019-01-23 2019-03-13 British American Tobacco Investments Ltd Method
US11213062B2 (en) 2019-05-09 2022-01-04 American Snuff Company Stabilizer for moist snuff
WO2023108162A2 (fr) * 2021-12-09 2023-06-15 Impello Biosciences, Inc. Procédés et compositions pour lutter contre des organismes nuisibles et des pathogènes de plantes
CN118044446A (zh) * 2024-04-01 2024-05-17 云南省烟草农业科学研究院 一种提高烤烟nc102成熟落黄的田间管理办法

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3593506A (en) * 1969-08-21 1971-07-20 Univ Kentucky Res Found Tobacco plant harvester
JPS5011965B2 (fr) * 1972-03-13 1975-05-08
DE3264786D1 (en) * 1981-11-04 1985-08-22 Basf Ag Acetylene derivatives, process for their preparation and their use in the regulation of the growth of plants
US4581057A (en) * 1984-03-15 1986-04-08 The Board Of Regents Of University Of Michigan, Corp. Of Michigan Abscisic acid containing foliar fertilizers and method of using same to enhance crop yields
DE3803667A1 (de) * 1988-02-06 1989-08-17 Basf Ag Mittel zur verminderung der transpiration von pflanzen
US5201931A (en) * 1988-12-01 1993-04-13 Her Majesty The Queen In Right Of Canada, As Represented By The National Research Council Of Canada Abscisic acid-related plant growth regulators - germination promoters
US5391568A (en) * 1992-07-10 1995-02-21 American Health Foundation Inhibition of lung tumorigenesis by administration of a polyphenol
FI98512C (fi) * 1995-06-09 1997-07-10 Cultor Oy Kasvien satotuloksen parantaminen
US5627132A (en) * 1995-11-07 1997-05-06 Voyager Enterprises Inc. Method and composition for plant preservation without leaf curling
US6138683A (en) * 1997-09-19 2000-10-31 Thione International, Inc. Smokeless tobacco products containing antioxidants
US5829449A (en) * 1997-09-19 1998-11-03 Thione International, Inc. Smoking products containing antioxidants
US6755200B1 (en) * 1999-11-19 2004-06-29 Philip Morris Incorporated Method for reduction of tobacco specific nitrosamines
US6564808B1 (en) * 2000-08-11 2003-05-20 Philip Morris Incorporated Method for reduction of tobacco specific nitrosamines
US20030056801A1 (en) * 2001-09-07 2003-03-27 Krauss Marc R. Method for the reduction of tobacco specific nitrosamines by increasing antioxidants in tobacco
AP2004003146A0 (en) * 2002-04-09 2004-09-30 Vector Tobacco Ltd Tobacco having reduced nicotine and nitrosamines
US20050034365A1 (en) * 2003-01-31 2005-02-17 Philip Morris Usa Inc. Spray and/or soil treatment of tobacco to reduce TSNAs

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7992575B2 (en) 2005-02-28 2011-08-09 U.S. Smokeless Tobacco Company Use of chlorate, sulfur or ozone to reduce tobacco specific nitrosamines
EP1951074A4 (fr) * 2005-11-07 2012-12-26 Swedish Match North Europe Ab Nicotine a teneur reduite en nitrosamines
WO2007053097A1 (fr) 2005-11-07 2007-05-10 Njette Ab Nicotine a teneur reduite en nitrosamines
US7757697B2 (en) * 2005-12-22 2010-07-20 Swisher International, Inc. Method for reducing nitrosamines in tobacco
US11547138B2 (en) 2007-09-07 2023-01-10 U.S. Smokeless Tobacco Company Llc Tobacco having reduced tobacco specific nitrosamine content
US10357055B2 (en) 2007-09-07 2019-07-23 U.S. Smokeless Tobacco Company Llc Tobacco having reduced tobacco specific nitrosamine content
US9271524B1 (en) 2007-09-07 2016-03-01 U.S. Smokeless Tobacco Company Tobacco having reduced tobacco specific nitrosamine content
EA021894B1 (ru) * 2009-10-09 2015-09-30 Филип Моррис Продактс С.А. Способ обработки табачного материала
WO2011042166A1 (fr) * 2009-10-09 2011-04-14 Philip Morris Products S.A. Traitement combiné d'un extrait de tabac utilisant des antioxydants et des épurateurs d'antioxydants
WO2013035505A1 (fr) * 2011-09-05 2013-03-14 日本たばこ産業株式会社 Procédé pour empêcher une augmentation des nitrosamines spécifiques au tabac pendant le stockage
JP2015527066A (ja) * 2012-08-08 2015-09-17 グルッポ モーロ サヴィオラ エス．アール．エル． ニトロソアミン含有量の低いタバコを製造するための方法
WO2014024020A1 (fr) * 2012-08-08 2014-02-13 Gruppo Mauro Saviola S.R.L. Procédé de fabrication d'un tabac à basses teneurs en nitrosamines
EP2981182A1 (fr) * 2013-04-05 2016-02-10 R. J. Reynolds Tobacco Company Modification du profil bactérien du tabac
CN104432463A (zh) * 2014-10-29 2015-03-25 湖北中烟工业有限责任公司 白肋烟加工醇化调香的方法
CN107920582A (zh) * 2015-05-14 2018-04-17 R.J.雷诺兹烟草公司 烟草的处理
CN112189882A (zh) * 2015-05-14 2021-01-08 R.J.雷诺兹烟草公司 烟草的处理
CN112189882B (zh) * 2015-05-14 2022-08-19 R.J.雷诺兹烟草公司 烟草的处理
CN105794477A (zh) * 2016-04-11 2016-07-27 广东省烟草南雄科学研究所 一种提高贫氯烟田烟叶氯离子含量的方法
CN109699650A (zh) * 2019-01-29 2019-05-03 中国热带农业科学院环境与植物保护研究所 一种高效诱导荔枝产生抗病性的组合物
CN111011032A (zh) * 2019-12-11 2020-04-17 中国烟草总公司郑州烟草研究院 一种降低氯敏感作物中氯离子含量的方法
CN111693650A (zh) * 2020-06-10 2020-09-22 河南中烟工业有限责任公司 一种改善烤烟上六片烟叶质量的方法
CN111693650B (zh) * 2020-06-10 2023-05-16 河南中烟工业有限责任公司 一种改善烤烟上六片烟叶质量的方法

Also Published As

Publication number Publication date
US8905041B2 (en) 2014-12-09
US20080202538A1 (en) 2008-08-28
US20130146073A1 (en) 2013-06-13
US20050034365A1 (en) 2005-02-17
WO2004068973A3 (fr) 2005-06-23
US8353300B2 (en) 2013-01-15

Similar Documents

Publication Publication Date Title
US8905041B2 (en) Sodium chlorine spray treatment of the leaves present on tobacco plants to significantly reduce TSNAs in cured tobacco produced therefrom
US20050072047A1 (en) Tobacco having reduced nicotine and nitrosamines
EP0110693B1 (fr) Mélanges de tabac pour remplisseur et articles à fumer contenant ces mélanges
US7293564B2 (en) Method for chemically modifying tobacco during curing
US7757697B2 (en) Method for reducing nitrosamines in tobacco
JP5976690B2 (ja) 乾燥処理されたタバコ及び乾燥処理方法
US20110289836A1 (en) Use of chlorate, sulfur or ozone to reduce tobacco specific nitrosamines
JP6210113B2 (ja) ニトロソアミン含有量の低いタバコを製造するための方法
US20060016125A1 (en) Light treatment for reduction of tobacco specific nitrosamines
US20030056801A1 (en) Method for the reduction of tobacco specific nitrosamines by increasing antioxidants in tobacco
CN104432465A (zh) 一种通过叶脉浸泡提高烟叶品质的烟叶调制方法
US20230107024A1 (en) Tobacco having reduced tobacco specific nitrosamine content
De Roton et al. Factors influencing the formation of tobacco-specific nitrosamines in French air-cured tobaccos in trials and at the farm level
CN109924530A (zh) 用于烟叶调制的提质减害调节剂及其使用方法
Dallmier et al. Effect of exogenous abscisic acid on proline dehydrogenase activity in maize (Zea mays L.)
CN109169124A (zh) 降低烤烟中亚硝胺含量的方法
US4127136A (en) Treatment of tobacco
CN113100471A (zh) 一种雪茄烟叶晾制提质减害调节剂及其使用方法
Li et al. Wounding of Root or Basal Stalk Prior to Harvest Affects Pre‐harvest Antioxidant Accumulation and Tobacco‐specific Nitrosamine Formation during Air Curing of Burley Tobacco (Nicotiana tabacum L.)
Sifola Quality characteristics of Burley tobacco irrigated with saline water
Crafts-Brandner et al. Effect of leaf maleic hydrazide concentration on yield and dry matter partitioning in burley tobacco (Nicotiana tabacum L.)
JP2025134221A (ja) ポリフェノール生合成促進方法
Goins Influence of ethylene gas on the chemistry and quality of underripe and ripe flue-cured tobacco.
Gupton et al. Some Effects of 2‐Chloroethyl‐phosphonic Acid (CEPA) on Burley Tobacco (Nicotiana tabacum L.) Cured Detached from the Stalk 1
Ogonowski et al. The Effect of Sucker Control Chemicals on the Yield, Quality and Composition of Flue-Cured Virginia Tobacco in Zambia: Part I: Effect on Growth and Yield

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase